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Preparation of Biodegradable Cationic Polycarbonates and Hydrogels Through the Direct Polymerization of Quaternized Cyclic Carbonates Alexander Y. Yuen, Elena Lopez Martinez, Enrique Gomez-Bengoa, Aitziber L. Cortajarena, Robert Hernandez Aguirresarobe, Amaury Bossion, David Mecerreyes, James L Hedrick, Yi Yan Yang, and Haritz Sardón ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00335 • Publication Date (Web): 28 Jun 2017 Downloaded from http://pubs.acs.org on July 2, 2017

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ACS Biomaterials Science & Engineering

Preparation of Biodegradable Cationic Polycarbonates and Hydrogels Through the Direct Polymerization of Quaternized Cyclic Carbonates Alexander Y. Yuena, Elena Lopez-Martinezc, Enrique Gomez-Bengoab, Aitziber L. Cortajarenac,e, Robert H. Aguirresarobea, Amaury Bossiona,d, David Mecerreyesa,e, James L. Hedrickf, Yi Yan Yangg, Haritz Sardona,e a

POLYMAT, University of the Basque Country UPV/EHU, Joxe Mari Korta Center, Avda. Tolosa 72, 20018 Donostia-San Sebastian, Spain. b

Departamento de Química Orgánica I, Facultad de Química, Universidad del País Vasco, Spain.

c

CIC biomaGUNE, Parque Tecnológico de San Sebastián, Paseo Miramón 182, Donostia-San Sebastián 20014, Spain.

d

e

f

University of Bordeaux, 351 Cours de la Liberation, 33400, Talence, France.

Ikerbasque, Basque Foundation for Science, E-48011 Bilbao, Spain.

IBM Almaden Research Center, 650 Harry Road, San Jose, CA 95120, USA.

g

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore

E-mail: [email protected]

ABSTRACT Polymers exhibiting both antimicrobial and biodegradable properties are of great interest for next generation materials in healthcare. Among those, cationic polycarbonates are one of the most promising classes of materials owing to their biodegradability, low toxicity and biocompatibility. They are typically prepared by a chemical post-modification after the polymer has been synthesized. The main problem with the latter is the challenges of ensuring and verifying complete quaternization within the polymer structure. Herein, we report the first example of synthesizing and polymerizing charged aliphatic cyclic carbonates with three different alkane pendant groups (N-methyl, N-butyl, and N-hexyl) by ring opening polymerization (ROP). These charged eight-membered cyclic carbonates displayed extraordinary reactivity and were even polymerizable in polar solvents (e.g., DMSO) and in catalyst free conditions that are generally unobtainable for other ring opening polymerization processes. A computational study was carried out and the findings were in agreement with the experimental data in regards to the dramatic increase in reactivity of the charged monomer over their neutral analogs. Furthermore, a series of hydrogels were prepared using the different charged eight-membered cyclic carbonates, and we found it to have a significant impact on the hydrogels’ ability to swell and degrade in water. Finally, the hydrogels demonstrated antibacterial activity against Escherichia 1 ACS Paragon Plus Environment


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coli (Gram-negative) and Staphylococcus aureus (Gram-positive). These materials could be ideal candidates for biologically relevant applications were the cationic structure is required. Keywords: Cyclic carbonates, antimicrobial, hydrogels, ring opening polymerization

2 ACS Paragon Plus Environment

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1. INTRODUCTION Polycations or cationic polymers are commonly employed nowadays in many different applications such as household products (e.g., soaps and shampoos)1 or flocculants for water purification2. Furthermore, they are being tested in biomedical applications such as drug delivery3,4, gene transfer5–9, and antimicrobial coatings10–19. Over the past decade, a considerable amount of attention in the scientific community has been given to the synthesis of cationic polymers with hydrolizable polymer backbones. The main reason is to develop cationic polymers which are biodegradable and do degrade after doing its job depending on the application in the environment or in the body. This is usually made possible by including urethane, ester, or carbonate functional groups in the polymers’ backbone, which can then readily hydrolyze under physiological conditions.8,20–30 Among those, cationic polycarbonates are one of the most promising class of materials owing to their fast biodegradability, low toxicity and biocompatibility.31 The current method of preparing biodegradable cationic polycarbonates is via post-chemical modifications made after the formation of the polymer. For example, Pascual et al., prepared cationic polymers via ring opening polymerization (ROP) of N-substituted eight-membered cyclic carbonates, which were then quaternized with methyl iodide.32 After this postquaternization step, the polymers exhibited broad-spectrum antimicrobial properties against gram-positive and gram-negative bacteria. Similarly, Hedrick et al. prepared polycarbonate based cationic polymers by first polymerizing cyclic carbonates containing alkyl chloride side chains, which were then post-functionalized with nitrogen containing reagents; the resulting cationic polymers were studied for antimicrobial applications.33 The main drawback to such a process lies in the difficulty in obtaining and ensuring complete quaternization.12,34,35 A more elegant method should rely on the ‘direct-polymerization’ of monomers already bearing quaternary ammonium functional groups. This approach ensures a better control of the degree of quaternization in the polymer.7,36 Upon evaluating the literature about the preparation of polycarbonates, they seem to be exclusively prepared via post-polymerization methods. We suspect this trend is due to the solubility of charged monomers in non-polar solvents. Generally, polar solvents are usually overlooked for use in ring opening polymerizations (ROP) because they tend to coordinate with active species and inhibit the ROP process. Therefore, only a few examples have been presented in the literature performing the ROP in highly polar solvents such as DMSO or DMF.37 In this work we investigate the ring-opening polymerization of cationic N-substituted eight-membered cyclic carbonates. The final goal is to develop a process to produce cationic polycarbonate hydrogels with antimicrobial properties by a direct-polymerization approach.



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2. RESULTS AND DISCUSSION We have recently demonstrated that N-substituted eight-membered cyclic carbonates posses superior reactivity in comparison to 5 and 6-membered cyclic carbonates. Consequently, we envision that these monomer families could be better suited to be polymerized in polar solvents rather than five and six-membered cyclic carbonates. In this work, a series of cationic monomers were prepared with different alkyl pendant chains (methyl, butyl, and hexyl) starting from Nsubstituted eight-membered cyclic carbonates. Afterwards, these monomers were homopolymerized and their reactivities were further studied with computational modeling. Hydrogels were prepared using the aforementioned monomers, and characterized with FTIR, water swelling, and rheology. Finally, the hydrogels were screened against both gram positive and gram negative bacteria to evaluate their use as antimicrobial agents.

2.1 Monomer synthesis and polymerization Non-charged eight-membered cyclic carbonates were synthesized using a ring closure process of diethanolamines with triphosgene, in the presence of triethylamine.32,38,39 Three cyclic monomers with different alkyl chains were prepared. Subsequently, these monomers were quaternized with iodomethane to afford the quaternized version of the eight-membered cyclic carbonates with three different alkyl chains (i.e. 8-Met, 8-But and 8-Hex) with excellent isolated yields of ~93% (Scheme 1). The monomer structure was confirmed by 1H and 13C NMR spectroscopy (SI section) Scheme 1. Charged aliphatic N-substituted eight-membered cyclic carbonates used in the direct preparation of cationic polycarbonates.

The ability to promote the polymerization of the functionalized monomer via ROP in the presence of DBU was evaluated (Scheme 2). The organocatalyst DBU was used here based on past successful polymerizations of eight-membered cyclic carbonates.38 The monomer 8-Met was insoluble in many organic solvents available except for DMSO. Although DMSO is not considered the most suitable solvent to perform ROP, a series of homopolymerizations with 8Met at three different degrees of polymerization (DPs) (i.e. 50, 100 and 200) were carried out (Table 1, Entries 3, 4, and 5).


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Scheme 2. Representative scheme to obtain linear homopolymers via ROP of monomer 8-Met, 8-But and 8-Hex.

Table 1. Exploration of homopolymerization of charged eight-membered cyclic monomers via ROP. Entry 1 2 3 4 5 6 7

Monomer Non-charged 8-Met

DBU amt

Time

% conv.

50

5.00 mol%

1 week

34 %

-

(none) 0.33 mol% 0.33 mol% 0.33 mol% 1.00 mol% 1.00 mol%

1d 1min 1min 1min 1min 1min

>97% >97% >97% >97% >97% >97%

14,200 14,900 20,600 39,300 64,000 71,200

50 8-Met 8-But 8-Hex

Mn by H NMR

Target DP

100 200 200 200

1

After screening reaction conditions with different catalyst loadings, we found that 0.33 mol% of DBU was optimum for the 8-Met monomer. It appeared that higher amounts of DBU allowed for different side reactions to take place (SI section). Using 1H NMR, the polymerizations were monitored by the disappearance of 8-Met’s methylene protons adjacent to the carbonate (4.62 ppm) and their subsequent reappearance at 4.59 ppm (Figure 3). Monomer conversion was determined using relative peak integrations values. Polymerization was also evidenced following the shift of the carbonyl by 13C NMR from 153.26 ppm to 152.92 ppm (SI section). Further characterization by FTIR-ATR provided additional evidence of the formation of polycarbonate by the disappearance of the monomer’s C=O stretching band at 1758 cm-1 and the appearance of a polycarbonate C=O band at 1751 cm-1. A large new peak at 1247 cm-1 was also observed and identified as the C-O from the polycarbonate (SI section).



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Figure 3. Homopolymerizations of 8-Met (DP: 50) were carried out and characterized with 1H NMR in d6-DMSO. Depicted above is of the homopolymer, and below is the monomer 8-Met. All polymerizations listed in Table 1 for the charged monomers at different DPs reached full conversion incredibly fast, especially in comparison to the non-charged monomer. For the analogous non-charged 8 monomer, we were only able to observe 34% monomer conversion, despite using much more DBU (5.00 mol%) and longer reaction time of one week in DMSO (Table 1, Entry 1). Moreover, we observed that 8-Met was reactive enough to polymerize by itself within 24 hrs without the presence of a catalyst, obtaining high molar masses calculated by 1 H NMR (Table 1, Entry 2). We attempted to characterize the polymers by SEC in various solvents (DMF, THF, and H2O) but we were unable to obtain any data. We suspect that the polymers had either (1) degraded into smaller chains or (2) got stuck in the column. We also attempted a post-polymerization anion exchange with LiTFSI in H2O. However, we were also unable to detect any presence of a polymer with our GPC-SEC setup. Polymerizations of 8-But and 8-Hex were carried out in a similar fashion as in the previous section, see Table 2. At first, 0.33 mol% DBU was applied in each polymerization and we observed a decrease in monomer conversion with increasing length of the alkane pendant group. As a solution, DBU content was increased to 1 mol% and we were again able to realize full monomer conversions of 8-But and 8-But in one minute reaction time. Polymerization were confirmed by 1H and 13C NMR (SI section). We also attempted to characterize the by SEC but we were unable to obtain any data even using THF and DMF with salts (ie: LiBr and LiTFSI) at different concentrations (5 and 10mM).


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The solubility of all the cationic polycarbonates was surveyed in a range of organic solvents (Table S1). The polymers were consistently insoluble in solvents such as DCM, ACN, THF, and acetone. All synthesized polycarbonates were soluble in polar aprotic solvents such as DMF and DMSO. The solubility in water was mainly governed by the alkyl chain length. All polymers derived from 8-Met were soluble in H2O, however the polymers loses its solubility in water as the pendant group is extended to 8-But and 8-Hex. From our homopolymerizations, we observed quite an extraordinary reactivity of the 8-Met and the other charged monomers. We observed that 8-Met was reactive enough to polymerize by itself within 24 hrs without the presence of a catalyst, obtaining high molar masses calculated by 1 H NMR (supporting information). Under the conditions used, complete monomer conversion of the noncharged 8-Met analog could not be obtained even when significantly more DBU was used. We have previously confirmed that the ring size matters in terms of reactivity40, but in this case a tremendous difference in terms of reactivity were found from charge to non-charge monomers in spite of the same ring size. Therefore, we decided to examine this system with a thorough computational study.

2.2 Computational modeling To gain a deeper insight into the reactivity of 8-Met, DFT studies were carried out1 with a special focus on the reasoning behind the high reactivity of the quaternary ammonium salts and the reactivity difference with their noncharged counterpart 8-Met. Also, the role of DBU in enhancing the reaction rate was studied. The calculations were performed at the M062X/6311+G(d,p) level with DMSO as the solvent (IEFPCM). Ammonium species A-1 served as models for the experimental substrate 8-Met. The neutral amine monomer A-2 was also computed, and the two substrates (A-1 and A-2) were taken as ground states (G=0) of their respective energy profiles. To understand the high reactivity of the ammonium salt 8-Met, we compared its energy profile with that of the neutral species A-2. Similar to our previous mechanistic study on a related system,2 the carbonate ring opening reaction was found to be a stepwise process (Figure 4). Thus, the initial nucleophilic addition of methanol to A-1 through TS1 leads to the formation of the quaternized intermediate (INT1), followed by a ring opening (TS2) process to the open-chain monomeric product (Figure 4). If the calculations are performed in the presence of a single explicit molecule of methanol40, the activation energies are exceedingly high and unaffordable (∆Gǂ = 41.7 kcal/mol), whilst the introduction of a second explicit molecule of methanol, like in Figure 4, seems critical for the correct solvation of the transition states, and thus, for the activation of the substrates, showing a large reduction of the activation barriers (TS1, ∆Gǂ = 21.0 kcal/mol). In fact, TS1 and TS2 are highly polar structures, with significant negative charge 1

The reported energy values correspond to Free energies (G) and are given in kcal/mol. For further computational details, see Supporting Information.



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development around the oxygen atoms of the carbonate, especially the carbonyl group, whose double bond is breaking during the initial attack. Therefore, the use of both explicit and implicit solvent systems seems to be determinant for the correct computational description of the systems, as well as for the experimental activation of the reaction. For A-1, the Gibbs Free energies of both steps are low and similar (TS1, ∆Gǂ = 21.0, TS2, ∆Gǂ = 20.7), contributing almost equally to the reaction rate. Me O H

H

-O

O

O + Me O

O + N

Me H O

O

G‡ = 21.0 TS1

O O

Me + N

Carbonate + 2 MeOH

+

O O H

Cl-

H

GR = -7.4

INT1 G = -2.5

O

Me

+ N Cl-

G‡ = 20.7 TS2

Cl-

-O

O

H O O

OMe O

A-1

MeO

O

+N Cl-

OH

+ MeOH

+ N Cl-

Figure 4. Reaction energy profile for substrate A-1 and its Gibbs Free energies computed at M062X/6-311+G(d,p) (iefpcm, solvent =DMSO) level. On the other hand, the comparison of the cationic species N+-Me2 species (A-1) vs the neutral NMe one (A-2) is also illustrative, since the former presents energy values in general 5-6 kcal/mol lower than the latter. This energy difference theoretically corresponds to a 104 times faster reaction rate, which is in fair agreement with the observed experimental rate increase for the ammonium salt. These findings confirm that the positive charge on nitrogen exerts a strong inductive effect through the carbon chain, making the remote carbonyl group highly electrophilic. Indeed, -+NR3 is known to present one of the strongest +I inductive effects. Finally, the role of DBU was also checked, finding that in its presence the activation energy of the process is reduced in ca. 10 kcal/mol, becoming a really fast process at ambient conditions, as found experimentally. DBU binds and activates the incoming molecule of methanol, increasing its nucleophilicity, during the initial attack TS1.


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O O

O N

Name TS1 INT1 TS2 Product

I

A-1

A-2

∆Gǂ = 21.0 ∆G = -2.5 ∆Gǂ = 20.7 ∆G = -7.4

∆Gǂ = 26.6 ∆G = 3.1 ∆Gǂ = 23.5 ∆G = -5.7

Figure 5. Comparison of the Gibbs Free energy values for the three different substrates, computed at M062X/6-311+G(d,p) (iefpcm, solvent =DMSO) level.

2.3 Charged polycarbonate hydrogels Due to the 3-D structure of hydrogels, it is difficult to ensure total quaternization of the entirety of the hydrogel using the post-quaternization method. Furthermore, it can be just as challenging to remove any unreacted quaternizing agent from the hydrogel. In order to prepare polycarbonate hydrogels, charged mono-functional eight-membered cyclic carbonates bearing were directly co-polymerized in DMSO with eight-membered dicyclic carbonate and PEG-diol, which served as a crosslinker and initiator, respectively. Again, DBU was employed to catalyze the ROP reactions (Figure 6A). The resulting hydrogels were named based on the charged eight-membered cyclic carbonate used in the synthesis (i.e. Gel-8-Met, Gel-8-But, and Gel-8-Hex). The reaction was confirmed by FTIR-ATR after the removal of DMSO from the gels. Evidence of the formation of polycarbonate structure was noted by the disappearance of the monomers’ C=O stretching band at 1758 cm-1 and the appearance of a C=O band at 1745 cm-1, considered to be the polymer. Furthermore, a large new peak at 1244 cm-1 was identified as the C-O from the polymer (Figure 6). To further confirm the formation of 3-D structures, the rheological behaviors of the gels at room temperature were also characterized using oscillatory tests (SI section). We were able to realize the elastic modulus (G’) to be greater than the viscous (G”) modulus for all the hydrogels. This G’ > G’’ behavior suggests that the gels that we obtained were covalently crosslinked. Furthermore, we continued to observe this behavior when we removed DMSO from one of the gels and re-swelling it in water (100 wt%).



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One particular characteristic of cationic hydrogels is their ability to swell in water without losing their three-dimensional structure due to their hydrophilicity. The level of swelling, responsiveness, and degradability are important features taken into account when designing these hydrogels. Thus, the swelling behaviors of the gels were studied by submerging dried pieces of gels in water. The “swelling behavior” was influenced by the ‘R’ pendant group of the functional monocyclic carbonate used to create the hydrogels. As expected, lengthening the alkyl chain from methyl to hexyl increases the hydrophobicity, and consequently its ability to swell water was reduced from 600 wt% to 55 wt% (Table S2). Aliphatic polycarbonates are known to rapidly hydrolize in the presence of water.32 Thus, the degradation in water at 25 °C of the gels with different alkyl chains was evaluated. The gels’ “time to degradation” was significantly prolonged from 8 h to 5 days by using monomers with longer alkane chains, (methyl < butyl

Preparation of Biodegradable Cationic ... - ACS Publications

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